Muscle-specific, liver-detargeted adeno-associated virus gene therapy rescues Pompe phenotype in adult and neonate Gaa-/- mice.

Pompe disease (PD) is a neuromuscular disorder caused by acid α-glucosidase (GAA) deficiency. Reduced GAA activity leads to pathological glycogen accumulation in cardiac and skeletal muscles responsible for severe heart impairment, respiratory defects, and muscle weakness. Enzyme replacement therapy with recombinant human GAA (rhGAA) is the standard-of-care treatment for PD, however, its efficacy is limited due to poor uptake in muscle and the development of an immune response. Multiple clinical trials are ongoing in PD with adeno-associated virus (AAV) vectors based on liver- and muscle-targeting. Current gene therapy approaches are limited by liver proliferation, poor muscle targeting, and the potential immune response to the hGAA transgene. To generate a treatment tailored to infantile-onset PD, we took advantage of a novel AAV capsid able to increase skeletal muscle targeting compared to AAV9 while reducing liver overload. When combined with a liver-muscle tandem promoter (LiMP), and despite the extensive liver-detargeting, this vector had a limited immune response to the hGAA transgene. This combination of capsid and promoter with improved muscle expression and specificity allowed for glycogen clearance in cardiac and skeletal muscles of Gaa-/- adult mice. In neonate Gaa-/- , complete rescue of glycogen content and muscle strength was observed 6 months after AAV vector injection. Our work highlights the importance of residual liver expression to control the immune response toward a potentially immunogenic transgene expressed in muscle. In conclusion, the demonstration of the efficacy of a muscle-specific AAV capsid-promoter combination for the full rescue of PD manifestation in both neonate and adult Gaa-/- provides a potential therapeutic avenue for the infantile-onset form of this devastating disease.

Gene therapy review: Duchenne muscular dystrophy case study.

Gene therapy, i.e., any therapeutic approach involving the use of genetic material as a drug and more largely altering the transcription or translation of one or more genes, covers a wide range of innovative methods for treating diseases, including neurological disorders. Although they share common principles, the numerous gene therapy approaches differ greatly in their mechanisms of action. They also differ in their maturity for some are already used in clinical practice while others have never been used in humans. The aim of this review is to present the whole range of gene therapy techniques through the example of Duchenne muscular dystrophy (DMD). DMD is a severe myopathy caused by mutations in the dystrophin gene leading to the lack of functional dystrophin protein. It is a disease known to all neurologists and in which almost all gene therapy methods were applied. Here we discuss the mechanisms of gene transfer techniques with or without viral vectors, DNA editing with or without matrix repair and those acting at the RNA level (RNA editing, exon skipping and STOP-codon readthrough). For each method, we present the results obtained in DMD with a particular focus on clinical data. This review aims also to outline the advantages, limitations and risks of gene therapy related to the approach used.

GRP78 clustering at the cell surface of neurons transduces the action of exogenous alpha-synuclein.

Mutation or multiplication of the alpha-synuclein (Syn)-encoding gene is frequent cause of early onset Parkinson's disease (PD). Recent evidences point to the pathogenic role of excess Syn also in sporadic PD. Syn is a cytosolic protein, which has been shown to be released from neurons. Here we provide evidence that extracellular Syn induces an increase in surface-exposed glucose-related protein of 78 kDa (GRP78), which becomes clustered in microdomains of the neuronal plasma membrane. Upon interacting with Syn, GRP78 activates a signaling cascade leading to cofilin 1 inactivation and stabilization of microfilaments, thus affecting morphology and dynamics of actin cytoskeleton in cultured neurons. Downregulation of GRP78 abolishes the activity of exogenous Syn, indicating that it is the primary target of Syn. Inactivation of cofilin 1 and stabilization of actin cytoskeleton are present also in fibroblasts derived from genetic PD patients, which show a dramatic increase in stress fibers. Similar changes are displayed by control cells incubated with the medium of PD fibroblasts, only when Syn is present. The accumulation of Syn in the extracellular milieu, its interaction with the plasma membrane and Syn-driven clustering of GRP78 appear, therefore, responsible for the dysregulation of actin turnover, leading to early deficits in synaptic function that precede neurodegeneration.

Selective disruption of the E-cadherin-catenin system by an algal toxin.

Yessotoxins (YTXs) are algal toxins that can be accumulated in edible molluscs. YTX treatment of MCF-7 breast cancer cells causes the accumulation of a 100 kDa fragment of E-cadherin, which we have named ECRA(100). A relative decrease in the concentrations of intact E-cadherin did not accompany the accumulation of ECRA(100) in cytosoluble extracts of MCF-7 cells on the first day of YTX treatment, but a collapse of the E-cadherin system was detected after 2-5 days of treatment with the toxin. An analysis of the general structure of ECRA(100) revealed that it consists of an E-cadherin fragment lacking the intracellular domain of the protein. ECRA(100) was not released into culture media of YTX-treated cells. Accumulation of ECRA(100) was observed in other epithelial cells, such as human intestine Caco-2 and MDCK cells after treatment with YTX. In turn, YTX could not induce accumulation of fragments of other members of the cadherin family, such as N-cadherin in the PC12 cell line and K-cadherin in sensitive cells (MCF-7, Caco-2, MDCK). The accumulation of a 100 kDa fragment of E-cadherin devoid of its intracellular domain induced by YTX was accompanied by reduced levels of beta- and gamma-catenins bound to E-cadherin, without a concomitant decrease in the total cytosoluble pools of beta- and gamma-catenins. Taken together, the results we obtained show that YTX causes the selective disruption of the E-cadherin-catenin system in epithelial cells, and raise some concern about the potential that an algal toxin found in seafood might disrupt the tumour suppressive functions of E-cadherin.